The present invention relates to the stabilization of labile enzymes in solutions. In a particular aspect, the present invention relates to soluble stabilized enzymes which can be useful in clinical diagnostic assays.
Enzymes are large molecular weight complex protein molecules, usually of unknown chemical structure. They are presently classified by their catalytic activity and extreme substrate specificity. Enzymes may be redefined as biological catalysts, capable of catalyzing a reaction of a single substrate or a reaction of a similar group of substrates.
Stability of enzymic solutions used in diagnostic assays is important in providing methods of analysis which exhibit precision and uniformity among separate determinations when conducted over a period of elapsed time. Instability of enzymic solutions, in addition to not providing reproducibility of assays, can also add to the ever increasing cost of medical services because the unstable enzymic solutions need to be discarded and fresh solutions formulated.
It has recently been estimated that about 25 percent of all in vitro diagnostic tests conducted annually in the United States are unreliable. Unreliable tests can result in unnecessary medical treatment, the withholding of necessary treatment and lost income. Because of their high specificity, the use of enzyme determinations has significantly increased during the last few years and indications are that this trend will continue. However, rigorous quality control measures are required to assure the accuracy and consistency of results. This requirement derives from the fact that the exact nature of enzymes, as well as mechanisms of their reactions remains unknown for the most part.
At present, the greatest limitation in the diagnostic reagent manufacture, by far, lies in the unstable characteristics of the enzymic solutions. Current diagnostic methodologies require the use of labile ingredients. Due to the labile nature of the enzymes, rigorous quality control is required over the production of such enzymic solutions, in the reconstituting dry media preparations and formulation of such enzymic solutions. Such quality control is costly. Moreover, if such control in any step in the process is not maintained within a high degree of precision, the quality of the final product can be reduced materially leading to the decreased precision in assay results.
The present commercial state of the art used for stabilizing the reactive ability of enzymes is by locking them into a solid matrix, either by freeze drying, dry blending such as used for tableting dry powders primarily in the pharmaceutical, diagnostic and related industries, and immobilization by locking the chemical structure of the enzyme into a solid matrix. Contrary to the sophistication these terms imply, these approaches are neither practical nor desirable and are also expensive. The manufacturer is forced to remove the water and supply a partial product, thus relinquishing part of the quality control cycle in the dilution and use of the final product. Laboratories are forced to pay the high cost of packaging, reagent waste, freeze drying and dry blending. Usefulness of the product is further limited by packaging modes and sizes.
Furthermore, good product uniformity is difficult to achieve, especially in the laboratories where the products are to be utilized in diagnostic assay. Generally, the reconstituted freeze-dried solutions have a relatively short stability such as about 24 hours to 5 days at room temperature conditions. Their use is then limited by such a short shelf-life.
The present invention is uniquely designed so that the enzyme solutions, although containing labile ingredients in a liquid regent, are effectively "stabilized" thereby controlling the activity of the labile ingredients in the liquid solution. This method of providing stability insures long-term stability in a liquid media. Moreover, close tolerance control can be achieved in the manufacturing of a high quality product which eliminates the inconvenience of the rigid package size, the high cost of packaging and freeze drying, and reagent waste.
In the clinical diagnostic field the commercial application of enzymic analysis is represented by, but not limited to, the diagnostic reagents used to determine and quantitate the following constituents in biological fluids;
1. glumatic-oxalacetic transaminase (SGOT); PA1 2. glutamic-pyruvic transaminase (SGPT); PA1 3. lactic dehydrogenase (LDH); PA1 4. creatine phosphokinase (CPK); PA1 5. .alpha.-hydroxybuteric dehydrogenase (.alpha.-HBD); PA1 6. glucose (via hexokinase-G-6-PDH or glucose dehydrogenase); and PA1 7. blood urea nitrogen (BUN). PA1 CP=Creatine phosphate PA1 ADP=Adenosine-5'-diphosphate PA1 ATP=Adenosine triphosphate PA1 HK=Hexokinase PA1 NAD=nicotinamide-adenine dinucleotide PA1 NADH=nicotinamide-adenine dinucleotide, reduced PA1 G-6-PDH=Glucose-6-phosphate dehydrogenase PA1 INT=tetrazolium salt PA1 PMS=phenazine methosulfate PA1 L-ASP=L-aspartic acid PA1 OAA=oxalacetic acid PA1 GLU=glutamic acid
The reagents for performing the diagnostic analyses for the above constituents react similarly, contain some common labile ingredients, and some of the chemical reactions involved are common. The following Reaction Scheme I is presented as a model to illustrate the general nature of the reactions involved: